Localization of plasminogen and plasminogen activators on cell surfaces arms cells with the proteolytic activity of plasmin. Cell surface proteolysis by plasmin is an essential feature of physiological and pathological processes requiring extracellular matrix degradation for cell migration 2,3, notably macrophage recruitment during the inflammatory response 4, as well as tissue remodeling 5, wound healing 6,7, tumor cell invasion and metastasis 8 and skeletal myogenesis 9, neurite outgrowth 10 and apoptosis 11. Furthermore, localization of plasminogen on the cell surface is required to facilitate macrophage recruitment in vivo 12. However, the specific molecules that account for the increased plasminogen binding capacity of the cells following differentiation to macrophages have not been elucidated.
Proteins exposing carboxyl-terminal lysines on cell surfaces are responsible for the ability of eukaryotic cells to bind plasminogen and enhance plasminogen activation because carboxypeptidase B (CpB) only partially reduces the plasminogen binding capacity of cells but completely blocks the cell-dependent stimulation of plasminogen activation 13. Thus, a specific subset of plasminogen binding sites, with carboxyl terminal lysines, is entirely responsible for the ability of cells to promote plasminogen activation. [These binding sites also interact with tissue plasminogen activator 14, lipoprotein(a) 15 and plasminogen fragments such as angiostatin 1.] Also in vivo results show that CpB-sensitive plasminogen receptors mediate monocyte recruitment in response to inflammatory stimuli 12. Therefore, the ideal candidate for a plasmin(ogen) receptor is an integral membrane protein that exposes a carboxyl terminal lysine on the cell surface. Such a protein has not been identified, to our knowledge.
A number of cell surface proteins with carboxyl terminal lysines have been identified as plasminogen binding proteins on cell surfaces of a variety of cell types 16. However, most proteins that have been identified do not have signal sequences and are known to be expressed intracellularly. No integral membrane protein with a carboxyl terminal lysine has been identified as a plasminogen receptor. A major difficulty in the field is that isolation and characterizations have been performed using SDS gels that are not ideal for resolution of integral membrane proteins.
Therefore, in order to search for an integral membrane protein with a carboxyl terminal lysine we employed a novel purification procedure followed by multidimensional protein identification technology (MudPIT). Intact cells were biotinylated and then either untreated or treated with CpB (to remove carboxyl terminal lysines). Then membrane fractions were prepared and isolated by affinity chromatography on plasminogen-Sepharose. The proteins that bound specifically to plasminogen-Sepharose were then captured on avidin-Sepharose, digested with trypsin and subjected to multidimensional protein identification technology (MudPIT). (Proteins that were not detected following treatment of intact cells with CpB were plasminogen binding proteins exposing C-terminal basic residues on the cell surface.) We identified a novel protein, C9orf46 homolog, that is predicted to be a Type II transmembrane protein that exposes a carboxyl terminal lysine on the cell surface, the first such candidate plasminogen receptor with this structure. We named the protein Plg-RKT, to refer to murine, human and all other orthologs. Furthermore, Plg-RKT expression was markedly upregulated when progenitor monocytes were differentiated with macrophage colony stimulating factor.
Our identification of peptides corresponding to Plg-RKT is, to our knowledge, the first demonstration of the existence of a protein encoded by the C9orf46 homolog gene present in the murine genome. The C9orf46 homolog DNA sequence encodes a protein of 147 amino acids with a calculated molecular weight of 17,261 Da. Notably, a carboxyl terminal lysine is present, consistent with the CpB sensitivity of this protein (on intact cells) in our isolation method and consistent with Plg-RKT as a candidate profibrinolytic plasminogen receptor. The Plg-RKT sequence is predicted to be a Type II (multipass) transmembrane protein with two predicted transmembrane domains from F53-L73 and P78—Y99. Hence, a 27 amino acid carboxyl terminal tail with a carboxyl terminal lysine is predicted to be exposed on the cell surface, again, consistent with our identification method, and placing the carboxyl terminal lysine in an orientation to bind plasminogen on the cell surface. We blasted the C9orf46 homolog/Plg-RKT sequence against all species using NCBI Blast and obtained unique human, rat, dog and cow orthologs, with high homology (e.g. human versus mouse=96% homology), high identity and no gaps in the sequence. Of key importance, a C-terminal lysine is predicted for all of the mammalian orthologs obtained in the blast search. In a query of the Ensembl Gene Report, DNA sequences of all other available mammalian orthologs (armadillo, lesser Madagascar hedgehog, rhesus monkey, gray short tailed opossom, domestic rabbit and chimpanzee) encoded C-terminal lysines, supporting functional importance of this residue.
The C9orf46 homolog/Plg-RKT transcript is broadly expressed in normal human and mouse tissues, [as determined using high-throughput gene expression profiling in which RNA samples from human and murine tissues were hybridized to high-density gene expression arrays 17,18] including spleen, thymus, lymph node, lung, intestine, bone marrow, as well as endocrine tissue, adrenal, pituitary vascular tissue, kidney, liver, stomach, bladder, and neuronal tissue (hippocampus, hypothalamus, cerebellum, cerebral cortex, olfactory bulb and dorsal root ganglion).
We searched for C9orf46 homolog/Plg-RKT mRNA microarray expression data in the European Bioinformatics Institute ArrayExpress Archive database. C9orf46 homolog mRNA is present in monocytes, leukocytes, NK cells, T cells, myeloid, dendritic, and plasmacytoid cells, breast cancer, acute lymphoblastic leukemia and Molt-4 acute lymphoblastic leukemia cells. These data are consistent with previous reports documenting expression of plasminogen binding sites on peripheral blood leukocytes19, breast cancer cells8,20 and other tissues [reviewed in 16]. In addition, results obtained by searching the ArrayExpress Warehouse indicated that the C9orf46 homolog gene is also regulated in other tissues by lipopolysaccharide, aldosterone, canrenoate, H2O2, and dexaamethasone. The broad distribution and regulation in tissues that express plasminogen binding sites, suggest that Plg-RKT provides plasminogen receptor function that may serve to modulate plasmin proteolytic functions in these tissues, as well. In genome-scale quantitative image analysis, overexpression of more than 86 cDNAs, including C9orf46 homolog, conferred dramatic increases in cell proliferation, while knockdown of C9orf46 homolog mRNA resulted in apoptosis21. In microarray studies, C9orf46 homolog mRNA expression has a high power to predict cervical lymph node metastasis in oral squamous cell carcinoma.
It is likely that Plg-RKT has not been identified previously because, being a membrane protein, it did not resolve well in SDS gel electrophoresis, the technique that has been used predominantly in the plasminogen receptor field for protein discovery. Furthermore, Plg-RKT represents the first plasminogen-binding protein that is a Type II membrane protein with a carboxyl terminal lysine on the extracellular face of the membrane in an orientation available to interact with plasminogen and in a location that can serve to localize plasminogen and plasmin to the cell surface.